Project Summary/Abstract: Bacterial infections are of serious concern to human health because of the continued emergence and spread of antibiotic resistance. Reports by both the Centers for Disease Control and World Health Organization have stated that the end of the antibiotic era is upon us. They also called for the development of new therapeutic strategies for treating bacterial pathogens, such as Staphylococcus aureus. A powerful strategy utilized by the host to combat invading pathogens is the restriction of essential nutrients, such as manganese (Mn) and zinc (Zn). Despite experiencing Mn and Zn starvation during infection, S. aureus and other successful pathogens remain capable of causing disease. Elucidating how bacteria adapt to this host defense has the potential to identify new targets for therapeutic intervention, the disruption of which will enhance the efficacy of the host immune response. Recent work revealed that a critical component of the nutrient withholding response is the Mn- and Zn-binding immune effector protein calprotectin (CP). This finding allowed the creation of novel reagents based on CP that can be used to impose highly specific and biologically relevant metal starvation in culture. Utilizing these reagents revealed that the bacterial two-component signal transduction system ArlRS is a critical regulator of the staphylococcal response to host-imposed metal starvation. Surprisingly, while ArlRS is known to contribute to staphylococcal virulence, the signal that activates the system and the direct vs. indirect targets of the system are unknown. Further investigations suggest that Mn and Zn sequestration prevents S. aureus from utilizing sugars, but not amino acids, as an energy source. This observation suggests that Mn and Zn starvation disrupts glycolysis in S. aureus. Recent work in other species has shown that glycolysis can be dependent on these metals. They also revealed that the expression of two putatively Mn-dependent superoxide dismutases, which is unique to S. aureus, promotes resistance to host- imposed nutrient metal starvation. In total, these results lead to the hypothesis that S. aureus profoundly alters how it generates energy and expresses alternative enzymes in order to adapt to host-imposed Mn and Zn starvation. The Aims of this proposal will evaluate this hypothesis and elucidate how S. aureus adapts to Mn and Zn starvation. Aim I. Elucidate the direct vs. indirect targets of the ArlRS regulatory network and the environmental signals that modulate activity of the system. Aim II. Determine the impact of Mn and Zn starvation on staphylococcal central metabolism and carbon source preference. Aim III. Elucidate how metal availability impacts the contributions of SodA and SodM to staphylococcal disease. To accomplish these aims an interdisciplinary approach utilizing techniques taken from microbiology and biochemistry, as well as advanced elemental quantification and whole cell paramagnetic spectroscopy, will be utilized.